Hepatitis C virus (HCV) is a global health problem, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Nearly 50% of infected patients do not respond to interferon therapy and it is crucial to develop additional therapeutic modalities. Hepatic injury is likely initiated by oxidants such as superoxide and peroxide that arise during viral infection and damage hepatocytes and surrounding cells. Hepatocytes normally up- regulate oxidative defense enzymes to maintain redox balance and prevent liver injury. Heme oxygenase-1 (HO-1) is an oxidative defense enzyme that is induced in response to oxidative stress. The enzyme's reaction products, biliverdin, carbon monoxide, and iron serve important functions in the cell to maintain cellular hemostasis and prevent injury. Past work from our laboratory has shown that HCV can transcriptionally regulate expression of HO-1. More recently, we reported that HO-1 overexpression or induction with hemin can attenuate viral replication and at the same time protect against oxidative injury in HCV replicon cells. Consequently, our findings strongly suggest that induction of HO-1 with drugs such as hemin may be a new exciting avenue for adjunctive antiviral therapy to treat HCV infection and/or prevent chronic liver disease. The major objective of this application is to characterize the cellular mechanisms whereby HO-1 induction and its enzymatic products inhibit HCV replication and reduce oxidative stress. The central hypothesis of this application is that HO-1 is an important hepatocellular defense enzyme whose reaction products attenuate HCV replication as well as protect against oxidative injury caused by the virus. Using established cell lines of HCV nonstructural and full length replicons as well as JFH6 line that supports the cellular life cycle of HCV in vitro, we will test the central hypothesis and accomplish the major objectives by undertaking experiments of three specific objectives: 1) We will investigate the role of HO reaction products iron and biliverdin/bilirubin as mediators of HO-1 antiviral activity in HCV infected hepatocytes. 2) We will investigate the role of heme oxygenase reaction products, biliverdin/bilirubin, carbon monoxide, and iron in reducing and/or modulating oxidative stress due to the virus, and 3) We will investigate the role of HCV as a regulator of HO-1 expression, under basal conditions and in response to stress. Since HO-1 is induced in response to oxidative stress it is important to clarify how the virus must modulate HO-1 expression. This work will expand our knowledge of viral replication and hepatic injury that occur during chronic HCV infection. It will also lay a foundation for development of a novel class of antiviral agents that potentially could be adjunctive therapy for chronic HCV infection. Potential Impact on Veterans healthcare: Veterans have higher incidence rates of chronic HCV infection and suffer increased morbidity and mortality than the population at large. The results of these studies are highly likely to positively impact veterans' care with new treatment modalities and management options. PUBLIC HEALTH RELEVANCE: Hepatitis C virus is a global health problem. This work will study how the virus causes human liver disease and how the human liver responds to the virus. The importance of this work is that it will be directly applicable to treatment of humans with hepatitis C virus infection.